Combining germline, tissue and liquid biopsy analysis by comprehensive genomic profiling to improve the yield of actionable variants in a real-world cancer cohort.

BACKGROUND: Comprehensive next-generation sequencing is widely used for precision oncology and precision prevention approaches. We aimed to determine the yield of actionable gene variants, the capacity to uncover hereditary predisposition and liquid biopsy appropriateness instead of, or in addition to, tumor tissue analysis, in a real-world cohort of cancer patients, who may benefit the most from comprehensive genomic profiling. METHODS: Seventy-eight matched germline/tumor tissue/liquid biopsy DNA and RNA samples were profiled using the Hereditary Cancer Panel (germline) and the TruSight Oncology 500 panel (tumor tissue/cfDNA) from 23 patients consecutively enrolled at our center according to at least one of the following criteria: no available therapeutic options; long responding patients potentially fit for other therapies; rare tumor; suspected hereditary cancer; primary cancer with high metastatic potential; tumor of unknown primary origin. Variants were annotated for OncoKB and AMP/ASCO/CAP classification. RESULTS: The overall yield of actionable somatic and germline variants was 57% (13/23 patients), and 43.5%, excluding variants previously identified by somatic or germline routine testing. The accuracy of tumor/cfDNA germline-focused analysis was demonstrated by overlapping results of germline testing. Five germline variants in BRCA1, VHL, CHEK1, ATM genes would have been missed without extended genomic profiling. A previously undetected BRAF p.V600E mutation was emblematic of the clinical utility of this approach in a patient with a liver undifferentiated embryonal sarcoma responsive to BRAF/MEK inhibition. CONCLUSIONS: Our study confirms the clinical relevance of performing extended parallel tumor DNA and cfDNA testing to broaden therapeutic options, to longitudinally monitor cfDNA during patient treatment, and to uncover possible hereditary predisposition following tumor sequencing in patient care.

Evaluating mismatch repair deficiency in colorectal cancer biopsy specimens.

Mismatch repair (MMR) testing on all new cases of colorectal cancer (CRC) has customarily been preferably performed on surgical specimens, as more tissue is available; however, new clinical trials for the use of immune checkpoint inhibitors in the neoadjuvant setting require MMR testing on biopsy samples. This study aims at identifying advantages, disadvantages and any potential pitfalls in MMR evaluation on biopsy tissue and how to cope with them. The study is prospective-retrospective, recruiting 141 biopsies (86 proficient (p)MMR and 55 deficient (d)MMR) and 97 paired surgical specimens (48 pMMR; 49 dMMR). In biopsy specimens, a high number of indeterminate stains was observed, in particular for MLH1 (31 cases, 56.4%). The main reasons were a punctate nuclear expression of MLH1, relatively weak MLH1 nuclear expression compared to internal controls, or both (making MLH1 loss difficult to interpret), which was solved by reducing primary incubation times for MLH1. A mean of  ≥ 5 biopsies had adequate immunostains, compared to ≤ 3 biopsies in inadequate cases. Conversely, surgical specimens rarely suffered from indeterminate reactions, while weaker staining intensity (p < 0.007) for MLH1 and PMS2 and increased patchiness grade (p < 0.0001) were seen. Central artefacts were almost exclusive to surgical specimens. MMR status classification was possible in 92/97 matched biopsy/resection specimen cases, and all of these were concordant (47 pMMR and 45 dMMR). Evaluation of MMR status on CRC biopsy samples is feasible, if pitfalls in interpretation are known, making laboratory-specific appropriate staining protocols fundamental for high-quality diagnoses.

Anti-tumoral effects of somatostatin analogs: a lesson from the CLARINET study.

Octreotide and lanreotide, the first-generation somatostatin analogs, successfully control hormone hyperproduction, and related syndromes, in patients with acromegaly and neuroendocrine tumors. However, their anti-tumor effect, rather evident in large number of pituitary adenomas in acromegalic patients, has been hypothesized for a long time in patients with neuroendocrine tumors as well, although a significant tumor shrinkage has rarely been observed. However, the recent publication of the CLARINET study has strengthened the evidence, already emerged with the PROMID trial, that the long-term treatment with the first-generation long-acting somatostatin analogs may exert an anti-tumor activity on G1 and G2 enteropancreatic neuroendocrine tumors, as well. After the publication, majority of international guidelines have updated their algorithms in line with these results and this class of drugs obtained the indication as anti-tumor agents in the majority of patients with neuroendocrine tumors.

Two doses of NGR-hTNF in combination with capecitabine plus oxaliplatin in colorectal cancer patients failing standard therapies.

BACKGROUND: asparagine-glycine-arginine-human tumour necrosis factor (NGR-hTNF), an agent selectively damaging the tumour vasculature, showed a biphasic dose-response curve in preclinical models. Previous phase I trials of NGR-hTNF indicated 0.8 and 45 µg/m(2) as optimal biological and maximum-tolerated dose, respectively. PATIENTS AND METHODS: Two sequential cohorts of 12 colorectal cancer (CRC) patients who had failed standard therapies received NGR-hTNF 0.8 or 45 µg/m(2) in combination with capecitabine-oxaliplatin (XELOX). RESULTS: Median number of prior treatment lines was 3 in the low-dose and 2 in the high-dose cohort. Overall, 21 patients had been pretreated with oxaliplatin-based regimens. No grade 3-4 NGR-hTNF-related toxicities were observed. Grade 1-2 chills were reported in 43% and 40% of cycles in the low-dose and high-dose cohorts, respectively. In the low-dose cohort, one patient achieved a partial response and five had stable disease for a median of 4.6 months. In the high-dose cohort, six patients had stable disease for a median of 3.6 months. Three-month progression-free survival (PFS) rates were 50% and 33% in the low-dose and high-dose cohort, respectively. Three patients in low-dose cohort experienced PFS longer than PFS on last prior therapy. CONCLUSIONS: Both NGR-hTNF doses were safely combined with XELOX in pretreated CRC patients. Hint of activity was apparent only with low-dose NGR-hTNF.

Possible protective role of metformin therapy on colonic polyps in acromegaly: an exploratory cross-sectional study.

CONTEXT: Colonic polyps occur in 30-40% of acromegalic patients, increasing the risk of colon carcinoma. Although debated, there is emerging evidence that metformin may play a protective role in diabetic and non-diabetic patients with colonic polyps and its use in chemoprevention is currently explored. OBJECTIVE: Evaluate the prevalence of colonic polyps in acromegalic patients treated or not with metformin and explore its possible protective role. DESIGN: Exploratory cross-sectional study in two tertiary Italian referral centres. MET: hods: Out of 153 acromegalic patients, we selected 58 patients (36-82 years; f: 33) who had at least one colonoscopy performed within the first 2 years of diagnosis. Presence of colonic polyps/cancer and related risk factors, current metformin and acetylsalicylic acid intake, disease duration, therapies for acromegaly, hormonal and metabolic parameters were assessed. RESULTS: An overall prevalence of 36% polyps was found. Based on the presence of polyps, we identified two groups, comparable for age, BMI, disease duration, glucose, insulin, HOMA-IR, HbA1c, GH and IGF-I levels. Of the patients with polyps (including three adenocarcinomas) only 24% were treated with metformin vs 57% of patients without polyps. Multivariate analysis confirmed a significant negative association between colonic polyps and metformin intake (OR: 0.22, 95% CI: 0.06-0.77, P = 0.01), whereas no significant association was found between polyps and age (P = 0.10), overweight/obesity (P = 0.54), smoking (P = 0.15), acetylsalicylic acid intake (P = 0.99), disease duration (P = 0.96), somatostatin analogues treatment (P = 0.70). CONCLUSIONS: These findings, though deriving from an exploratory study, could suggest a protective role of metformin on the development of colonic polyps in acromegaly, and need to be confirmed in an extended study population.

First-line single-agent cetuximab in patients with advanced colorectal cancer.

BACKGROUND: The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is active in heavily pretreated patients with metastatic colorectal cancer (mCRC) both in monotherapy and in combination with chemotherapy (CT). This study assesses the antitumor activity of single-agent cetuximab in CT-naive patients. PATIENTS AND METHODS: Phase II clinical trial was used. Patients were EGFR positive by immunohistochemistry and were not candidate for radical surgery, even in the case of substantial tumor shrinkage. Cetuximab was administered weekly. RESULTS: Thirty-nine patients were treated and evaluated. The most common adverse event was skin toxicity (89% any grade; 48% grade 1; 31% grade 2; 10% grade 3). One patient had a complete response and three obtained partial responses (10% overall response rate). Thirteen patients had stable disease (34%). Twenty-two patients experienced progressive disease (56%). Overall median time to progression (TTP) was 2 months, and the responders individual TTP was 12, 9, 9, and 6 months. CONCLUSIONS: Even in chemo-naive patients, cetuximab as single agent is active only in a small fraction of mCRC, similarly to what has been reported for heavily pretreated patients. The extent of benefit when response occurs is, however, such that it is mandatory to intensify the search for the predictive markers of response to cetuximab therapy.

Association of APC gene mutations and histological characteristics of colorectal adenomas.

Fifty-nine colonic adenomas and 6 hyperplastic colonic polyps were analyzed by single-strand conformation polymorphism analysis for mutations in the adenomatous polyposis coli gene (APC). Frameshifts and premature stop codons in at least one copy of APC were detected in 25 of these adenomas. Five adenomas carried 2 APC mutations. No mutations in APC were found in any of the 6 hyperplastic polyps. The detection of APC mutations increased with size and degree of dysplasia and in rectal as compared to colonic adenomas, although the association was not statistically significant. The frequency of detectable APC mutations was higher in tubulovillous and villous adenomas (10 of 13) than in tubular adenomas (15 of 45) (odds ratio, 6.67; 95% confidence limits, 1.39-41.83; P = 0.005). The significance of the association between the detection of APC mutations and a villous architecture was confirmed in multivariate analysis (relative risk, 6.67; 95% confidence limits, 1.54-28.8; P = 0.005). In conclusion, APC mutation plays a role in adenoma progression; its frequency is significantly higher in lesions with a more villous morphology.

Predictive value of rectal bleeding for distal colonic neoplastic lesions in a screened population.

The aim of this study was to determine the diagnostic value of rectal bleeding for distal colorectal cancer (CRC), or large (> or =10 mm) adenomas among an average-risk population. A cross-sectional survey was conducted among individuals aged 55-64 years, who attended sigmoidoscopy (FS) screening in the context of a multicentre randomised trial of FS screening for CRC. Sensitivity, specificity and positive predictive value (PPV) of rectal bleeding for large distal adenomas or CRC were calculated. Rectal bleeding was reported by 8.8% of 8507 patients examined (15% of those with large adenomas and 29% of those with CRC). The risk of CRC was increased when bleeding was associated with an altered bowel habit: odds ratio (OR)=10.42; 95% Confidence Interval (CI): 4.08-26.59; the corresponding OR for isolated bleeding was 5.29 (95% CI: 2.28-12.30). Rectal bleeding carries an increased risk of distal neoplastic lesions. However, most lesions are detected among asymptomatic subjects. This finding suggests that screening represents the optimal strategy to detect CRC or large adenomas in the distal colon in the targeted age range.

Genetic events in sporadic colorectal adenomas: K-ras and p53 heterozygous mutations are not sufficient for malignant progression.

Twenty-four sporadic colorectal adenomas were analysed for the presence of allelic loss on the short arm of chromosome 17 as well as mutations in the K-ras and p53 genes. Chromosome 17p13 allelic loss was not present in 14 out of 14 informative cases. K-ras mutations were observed in 15 out of 24 cases. A p53 gene mutation (GGC-->GAC at codon 245) was detected in two biopsies taken at a four year interval from a recurrent rectal villous adenoma. Both biopsies also contained the same K-ras gene mutation (GGT-->GTT at codon 12). The data from the recurrent rectal adenoma provide in vivo evidence that K-ras and p53 heterozygous mutations confer a proliferative advantage but together are not sufficient for malignant transformation.

Genomic PCR-SSCP analysis of the metastasis associated NM23-H1 (NME1) gene: a study on colorectal cancer.

To facilitate further mutational analysis of NM13-H1, a human metastasis suppressor gene, we have established its genomic organization. NM23-H1 is composed of five exons, spanning a genomic DNA fragment of 10 kb. Using oligonucleotide primers flanking each exon, PCR-SSCP analysis was performed on genomic DNAs of healthy individuals. A common polymorphism, a C to T transition, was detected 30 nucleotides upstream from the 5' splice site flanking exon 1. As NM23-H1 allele loss and altered expression have been reported in colorectal cancer, genomic DNAs of 20 colorectal tumors were analyzed for the presence of gene-specific mutations by PCR-SSCP: no abnormal sequences were detected within the coding and splice site regions of the NM23-H1 gene. This finding suggests that NM23-H1 mutations are rare events in human colorectal cancer.

Prevalence of the E1317Q variant of the APC gene in Italian patients with colorectal adenomas.

Loss of APC is an initial, rate-limiting event in inherited and sporadic colorectal tumorigenesis. Rare germline APC mutations have been identified in patients with multiple colorectal adenomas. Recently, the E1317Q APC variant has been associated with a predisposition to the development of multiple colorectal adenomas. In this study, the prevalence of the E1317Q variant was examined in 182 patients with single or multiple colorectal adenomas, and in 235 controls. In all, E1317Q was identified in two of 182 patients with adenomatous polyps (1.1%) and in two of 235 controls (0.8%) (p = 0.59). The risk of harboring adenoma(s) among subjects bearing the E1317Q variant was 1.29 (95% CI 0.09-18.0). No difference in the prevalence of E1317Q between cases with single (2.0%) or multiple colorectal adenomas (0.7%) and controls (0.8%) was found. None of the subjects with a family history of colorectal cancer carried the E1317Q variant. In conclusion, our results confirm that only a very small fraction of colorectal adenomas may be associated with the presence of E1317Q.

DNA flow cytometry of endoscopically examined colorectal adenocarcinomas.

The purpose of the study was to evaluate the correlation of DNA-ploidy of colorectal adenocarcinomas (adk) with histological and clinical parameters including the survival of the patients. Multiple biopsies from 95 adk were taken during colonoscopy prior to surgery. The samples were used to obtain nuclei suspensions for specific staining of DNA content and high resolution flow cytometry. DNA-aneuploidy, i.e. the presence of more than one G0/G1 peak, was detected in 67/95 cases (71%). The individual-specific control mucosa was DNA-diploid in all cases. The mean fraction of S-phase cells was 7.2% in control mucosa and 13.6% in adk. DNA-ploidy did neither correlate with Dukes' stage nor with differentiation degree. Among the patients studied for the correlation of DNA ploidy with survival for a period extending to 30 months (n = 51), the DNA aneuploid group was estimated to be about 5 times as risky as the DNA diploid group with respect to the odds of dying. We conclude that DNA flow cytometry of colorectal adk may predict clinical outcome and be helpful in addition to histopathology.

Contribution of germline mutations in the BRCA and PALB2 genes to pancreatic cancer in Italy.

Pancreatic adenocarcinoma (PC) is the third most common cancer associated with BRCA mutations. Most notice has been given to BRCA2, while the association between BRCA1 and PC is less widely reported. Recently, PALB2 has been implicated in both PC and breast cancer (BC) susceptibility. We selected 29 Italian PC patients from a case-control study of PC according to their personal and family history of both PC and breast/ovarian cancer (BC/OC) and tested them for presence of germline mutations in BRCA1, BRCA2 and PALB2. We identified no germline mutations or deletions in PALB2, but detected 7 BRCA mutations (4 in BRCA1 and 3 in BRCA2). These findings suggest that PALB2 does not play a major role in PC susceptibility in our population. As we found an almost equal frequency of germline mutations in BRCA1 and BRCA2, germline alterations in either of these genes may explain a subset of Italian families presenting both PC and BC/OC. Moreover, as we began the observation of these families from probands who are affected by PC, we provide here a direct assessment of the role of PALB2 and BRCA mutations in PC susceptibility.

Germline MLH1 and MSH2 mutations in Italian pancreatic cancer patients with suspected Lynch syndrome.

Lynch syndrome is an inherited cancer syndrome caused by germline mutations in mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. LS predisposes to high risk of early-onset colorectal, endometrial and other tumors. Patients with Lynch syndrome have also been shown to have an elevated risk for pancreatic cancer (PC). In this study, we aimed to estimate the frequency of suspected Lynch syndrome among a series of 135 PC patients. Further, we wanted to determine the frequency of MMR gene mutations in the suspected Lynch syndrome cases. We also aimed to verify the pathogenicity of any novel non-truncating variants we might detect with a functional assay. Based on personal and/or familial cancer history, 19 patients were classified as suspected Lynch syndrome cases. DNA material for mutation analysis was available for eleven of them. Four patients were found to carry a total of five MLH1 or MSH2 variants. Of these, MSH2-Q402X, MSH2-G322D, and MLH1-K618A had been previously reported, while the MSH2-E205Q and MSH2-V367I variants were novel. MSH2-Q402X is a known stop mutation and reported here for the first time here in association with PC. MLH1-K618A was found in the unaffected branch of a kindred, suggesting that it may be a polymorphism or a low penetrance variant. MSH2-G322D likely does not cause a MMR defect, although this variant has also been associated with breast cancer as indeed seen in our patient. The novel variants MSH2-E205Q and MSH2-V367I were found in the same patient. Both novel variants were however functional in the applied MMR assay. Our findings suggest that only a small subset of pancreatic cancer patients carry pathogenic MMR mutations.

The clinical significance of gastric epithelial dysplasia.

Six-hundred-and-ninety-four patients with localized gastric lesions (ulcers, polyps, enlarged or irregular folds, erosions and tumors) underwent endoscopic examination and multiple biopsies. Twenty-nine proved to have severe or moderate gastric epithelial dysplasia. Another group of 123 patients were submitted to endoscopic examination and multiple biopsies, but showed no evidence of localized gastric lesions. Only one case of mild dysplasia was observed in this group. Of the 29 patients with dysplasia, 8 (27.5%) had a carcinoma in the surgical specimen. The endoscopic follow-up carried out in non-operated patients with dysplasia did not show any development of malignancy. This study shows dysplastic changes significantly associated with prominent or depressed lesions of the gastric mucosa at endoscopy. In operated patients a synchronous carcinoma is often detected in the surgical specimen.

DNA flow cytometry of endoscopically examined colorectal adenomas and adenocarcinomas.

DNA ploidy of 64 colorectal adenomas and 49 adenocarcinomas, examined endoscopically, was studied by flow cytometry. We found DNA aneuploidy in none of the 105 normal mucosa samples (0%), in 20 adenomas (31%), and in 36 adenocarcinomas (74%). DNA ploidy of adenomas correlated with size (P = 0.02) and degree of dysplasia (P less than 0.01) but not with histologic type. Adenomas had a 45% incidence of DNA aneuploid stem lines in the DNA index range of 0.80-1.20, compared with 8% in the case of adenocarcinomas. The distribution of the DNA index values of adenocarcinomas was approximately normal, with a mean value 1.63 +/- 0.28. The mean DNA index for the three cases of "carcinoma in adenoma" with invasion of the stalk of the adenoma was 1.52 +/- 0.18. These results, using DNA flow cytometry, provide evidence for the progression of colorectal adenoma to adenocarcinoma. The classification of adenomas according to DNA ploidy may be information of considerable practical value to the clinician in predicting risk of further adenomas and/or risk of cancer.